Effect of anti-TNF/IL-6 NANOBODY® VHH, Humira, Sylvant, TNF, IL6 and TNF+IL6 in RA-FLS/T cell cocultures

Additive effects of TNF and IL-6 were evaluated in cell-based model for rheumatoid arthritis (RA) and then compared to treatment with anti-TNF/IL-6 NANOBODY® VHH, Humira and Sylvant by RNA-seq Overall design: Fibroblast-like synoviocytes from RA donors (RA-FLS, Cell Applications, Inc.) were cocultured with CD4 T cells from healthy human donors (n=9, 3 RA-FLS donors combined with 3 different CD4 T cell donors). PBMC's were isolated from whole blood using Ficoll-Paque (GE Healthcare). CD4 T cells were purified from PBMCs by negative selection (Milteny Biotec) and directly added to attached RA-FLS in a 10:1 ratio (CD4: RA-FLS). Additive effects of TNF and IL-6 were evaluated by stimulating RA-FLS/T cell coculture with recombinant TNF (Sigma), IL-6/sIL-6R (R&D) or a combination. Similar experiments were performed with clinically approved anti-TNF and anti-IL-6 antibodies (Humira and Sylvant, from pharmacy), their combination and our bispecific anti-TNF/IL-6 NANOBODY® VHH after stimulation with 100ng/ml recombinant IL-17A, sIL-6R (R&D) and anti-CD3 (Biolegend). For these experiments, antibodies were compared to IgG1 isotype control, while anti-TNF/IL-6 NANOBODY® VHH was compared to a NANOBODY isotype control.